Improved chromatographic separation of modified deoxycytidine nucleosides enhances LC-MS sensitivity

Cytosine methylation is an epigenetic modification that regulates transcription. DNA demethylation occurs through oxidation of methylcytosine to hydroxymethylcytosine, making hydroxymethylation an important and translationally relevant marker of DNA methylation dynamics. These epigenetic modifications can be measured in their deoxynucleoside forms from enzymatically digested DNA by liquid chromatography-mass spectrometry (LC-MS). However, 5-hydroxymethyl-2'-deoxycytidine (5hmdC) co-elutes with 2'-deoxycytidine (dC). Because 5hmdC represents <0.05% of total dC in DNA, co-elution with dC causes ion suppression that limits detection and quantitation. Here, we present a chromatographic method that separates 5hmdC from dC, eliminates ion suppression, and improves the sensitivity of 5hmdC detection.